Agglutination and Precipitation PDF

MLS 17a IMMUNOLOGY AND SEROLOGY LABORATORY MODULE 3: Secondary Serologic Reactions: Unlabeled 2ND SEMESTER...

MLS 17a IMMUNOLOGY AND SEROLOGY LABORATORY MODULE 3: Secondary Serologic Reactions: Unlabeled 2ND SEMESTER PRELIMS A.Y. 2023-2024 Immunoassay and Complement Fixation Test LECTURER: Matthew Tubola & Jeff Tolentino 🗣 📘 Legend: 💡 Turgeon 6th ED, Additional info, Recording 💻PPT, 💡 ADDITIONAL INFO INTRODUCTION Precipitation SOLUBLE ANTIGEN + SOLUBLE ANTIBODY = visible 💡 ADDITIONAL INFO INSOLUBLE COMPLEX Agglutination – involves a PARTICULATE ANTIGEN (bacteria, The precipitation and agglutination reactions were the first cells, or latex particles) which immunoassays developed and rely on the fact that aggregates to form a visible larger complex. antibodies and most antigens have multiple binding sites. Complement fixation – occurs after the binding of antigen The multiple binding sites of the antibodies and antigens and antibody, uptake of COMPLEMENT can be used as an result in the formation of large complexes when antigens indicator of the presence of specific antigen or antibody. and antibodies meet at appropriate concentrations. ❖ Secondary serologic reactions occur between antigens and antibodies when the antigens are 🗣 ADDITIONAL INFO multivalent and are most often visible. Precipitation and agglutination are the same, that is the antigen-antibody interaction and cross-linking. The 💡 ADDITIONAL INFO difference is the type of antigen that we use. In precipitation, soluble antigen is used, while in Secondary serologic reactions agglutination, particulate antigens are used. IN VITRO demonstrable antigen-antibody reaction. Occurs NOTE: between antigen-antibody when antigens are mostly Particulate antigen is bigger than soluble antigen. multivalent and are most often visible. Examples: Precipitation, Agglutination and Complement Latex particle is an example of a plastic polymer that is a Fixation (These are UNLABELED IMMUNOASSAYS) carrier molecule of an antigen. NOTE: Antibody + Univalent Antigen = Primary Serologic Reaction ❖ All serologic reactions, whether unlabeled or labeled, rely on the affinity, avidity, and specificity of Antibody + Multivalent Antigen = Secondary Serologic the antibody to bind to the antigen. Reaction Ag-Ab reaction In Vivo = Tertiary Serologic Reaction 💡 ADDITIONAL INFO Precipitation and agglutination reactions were the first The first immunoassay developed that followed this principle developed. Both are termed unlabeled immunoassays were the unlabeled immunoassays: because once there is antigen-antibody reaction, there is a formation of a visible result using the naked eye, and these PRECIPITATION - The cross-linking of a soluble results are called precipitation or agglutination. antigen to create an insoluble precipitate that is visible. To different precipitation from agglutination, look at the characteristics/nature of the ANTIGEN involved. AGGLUTINATION - The cross-linking of particulate antigens (bacteria, cells, or latex particles) to form Precipitation: Antibody + Soluble antigens = too small larger complexes that are also visible. antigens or floats in the serum. COMPLEMENT FIXATION TEST - Based on the fact Agglutination: Antibody + Particulate antigens = too big that when an antigen combines with an antibody in antigens the presence of complement, the complement is fixed by the antigen-antibody complex and is unable Complement fixation test, we observe the presence of to react with cells sensitized with other Ag-Ab reaction through the aid of complement proteins. antigen-antibody complexes. Ag + Ab + Complement Proteins Immunology and Serology Laboratory | Trans by: (Bayotas, S., Javier, M.K., Letsoncito, K.J.)  MLS 17a IMMUNOLOGY AND SEROLOGY LABORATORY MODULE 3: Secondary Serologic Reactions: Unlabeled 2ND SEMESTER PRELIMS A.Y. 2023-2024 Immunoassay and Complement Fixation Test LECTURER: Matthew Tubola & Jeff Tolentino AGGLUTINATION ❖ These factors can also be manipulated to remove an ❖ Agglutination is the process that occurs if an antigen antibody from an antigen (e.g. elution) 💡 is mixed with its corresponding antibody called isoagglutinin. ADDITIONAL INFO Agglutinin (soluble antibody) forms a lattice with an insoluble (particulate or Initial binding of any antibody to any antigen is called cellular) surface antigen. sensitization, regardless of antigen type (if red cell, etc.). ❖ The formation of clumps of cells or inert particles by There are many non-covalent bonds that stabilize the Ag-Ab specific antibodies to surface antigenic components interaction (hydrogen, ionic, Van der Waals, hydrophobic). (Direct agglutination) or to antigenic components adsorbed or chemically coupled to red cells or inert particles (Passive hemagglutination and passive Table: Factors Affecting Sensitization agglutination, respectively.) FACTORS DESCRIPTIONS 💡 ADDITIONAL INFO pH A pH of 6.5 to 7.5 is ideal for most antigen-antibody reactions while some antibodies AGGLUTINATION react stronger at a more acidic pH. ➔ It is when agglutinin (soluble antibody) forms a lattice with an insoluble (particulate or cellular) Temperature IgM isotypes react optimally at colder temperatures (e.g. room temperature), whereas surface antigen. ANTIGEN is mixed with its IgG isotypes react optimally at warmer corresponding ANTIBODY called ISOAGGLUTININ temperatures (e.g. 37 C). This is because the IgM antibody-carbohydrate 2 stages: 1. Sensitization 2. Lattice Formation and Opsonization antigen complex is held together through hydrogen bonds: hydrogen bonds are exothermic and have higher stability at colder temperatures. Notably, most IgM antibodies are directed against carbohydrate epitopes; this is because carbohydrates, unlike peptides, are ineffective at eliciting a T-helper cell response required for IgG class switching. Ionic A reduction in the ionic strength of the testing 💡 Strength medium can increase the rate of association between red cell antigens and antibodies. Low ADDITIONAL INFO ionic saline solution (LISS) can be used to take advantage of this property. Sensitization - initial binding - no visible agglutination Antibody - There should be an optimal proportion of Lattice formation and optimization - there is now Antigen ratio antibody and antigen to ensure equilibrium cross-linking - lattice can be visualized as an agglutination or between the rates of antibody association and agglomerate. dissociation. Antibody or antigen excess can disturb this equilibrium. NOTE: Lattice formation and optimization are combined in this Although the concentration of the red cells may not differ, the zygosity of the antigen may result in lesson since both have visible reactions in this stage. higher or lower quantities of the antigen on the red cell surface. Optimization = cross-lining of all the lattice formed SENSITIZATION 💡 ADDITIONAL INFO ❖ This involves the initial binding of antibodies to the red cell antigen through non-covalent bonds pH (of solution) ❖ The binding of a single antibody to a single red cell ➔ pH of 6.5 – 7.5 is the most ideal for antigen is not visible to the naked eye. Antigen-Antibody reactions. Slightly acidic to ❖ Several factors can affect the equilibrium constant of slightly alkaline. This is the optimum pH that we the antibody that dictates how strongly it binds to need to obtain in the sample in order to enhance the corresponding antigen. the sensitization of the Ab to the Ag. Immunology and Serology Laboratory | Trans by: (Bayotas, S., Javier, M.K., Letsoncito, K.J.)  MLS 17a IMMUNOLOGY AND SEROLOGY LABORATORY MODULE 3: Secondary Serologic Reactions: Unlabeled 2ND SEMESTER PRELIMS A.Y. 2023-2024 Immunoassay and Complement Fixation Test LECTURER: Matthew Tubola & Jeff Tolentino charged sialic acid on the red cell Temperature membrane. ➔ In Immunohema/BB, there are cold and warm ❖ In a saline solution, the zeta potential also depends reacting antibodies. on the ionic cloud that surrounds the red cells ➔ IgM - Cold reacting antibodies; IgG - Warm reacting antibodies Table: Factors Affecting Lattice Formation ➔ If IgG in nature, we follow the incubation step at 37 deg. Celsius to enhance sensitization or FACTORS DESCRIPTIONS binding of IgG to Ag. ➔ If IgM, incubation is not required since they react Zeta Potential RBCs are negatively-charged because of the at room temperature. presence of sialic acid. Because it is negatively-charged, it will attract positively-charged ions from Ionic Strength the solution that will create a sheer plane (gray ➔ The strength of the ions provided by the solution. area), as the zeta potential. Zeta potential is a ➔ For example, red blood cells are negatively barrier that will keep repulsing power against charged. The repulsion between red blood cells is another RBC. Elimination of reduction of the Zeta the zeta potential (the natural repulsion). Red potential makes one RBC closer to one another. blood cells do not attach with each other since Once RBCs are close to each other, IgG antibodies can easily bridge the gap between the two RBCs. they are negatively charged due to the sialic acid attached on the membrane of the cells. In the laboratory, we use saline composed of ions, and these ions will go to the center of the red blood cells forming the ionic cloud. ➔ The zeta potential is directly proportional to the ionic strength. The higher the ionic strength, the higher the zeta potential. ➔ With this, the space between the red blood cells Antibody Since most of the antibodies we are interested in is also higher. Here enters the low ionic strength Isotypes are IgG in nature, a specific reagent containing saline solution (LISS). The iconic strength of this IgG antibodies with specificity against the Fc or saline solution is low, and since the ionic strength constant region of other IgG molecules was is low, the ionic cloud is also not strong, the zeta developed. potential is also low. As a result, the red blood cells are also closer with each other or attach with each other, and it is now easy for the IgG antibody to cross link the two red blood cells. Antibody-Antigen Ratio ➔ We can see visible results once there is optimum proportion between the Ab and the Ag. It is okay if the Ag or Ab is slightly excess, or if they are not equal, however, when the excess is too much, then we cannot form agglutination. (haha kamot ni sir sabad) Prozone phenomenon - Excess antibody Agitation and The force produced through agitation or Post zone phenomenon - Excess antigen Centrifugation centrifugation can bring antibodies and antigens Zone of Equivalence - Reaction happens wherein the closer together in physical space. proportion of the Ab and Ag is equal. LATTICE FORMATION AND OPSONIZATION ❖ Once a red cell/inert particle is coated with antibodies, the binding of the antibodies to multiple red cells/inert particles causes visible red cell/inert Polymers Polymers, or potentiators, can enhance particle clumping to occur. agglutination through the reduction of the zeta ❖ The formation of a lattice requires the ability to potential. overcome the natural repulsion between two neighboring red cells. Both albumin and polyethylene glycol (PEG) are This electronic potential (or zeta thought to act by displacing water molecules in potential) is due to the negatively between red cells, which in turn decreases the Immunology and Serology Laboratory | Trans by: (Bayotas, S., Javier, M.K., Letsoncito, K.J.)  MLS 17a IMMUNOLOGY AND SEROLOGY LABORATORY MODULE 3: Secondary Serologic Reactions: Unlabeled 2ND SEMESTER PRELIMS A.Y. 2023-2024 Immunoassay and Complement Fixation Test LECTURER: Matthew Tubola & Jeff Tolentino zeta potential and allows the red blood cells to between two repelling red cells to allow for come into closer contact. agglutination. IgG only has a reach of 12-14 nm and is mostly unable to overcome the distance Proteolytic Enzymes can enhance agglutination by removing between two red cells. Enzymes the negatively charged sialic acid from the red cell ➔ Since most of the antibodies we are interested in membrane, which also reduces the zeta potential. are IgG in nature, a specific reagent containing Enzymes can also remove red cell glycoproteins IgG antibodies with specificity against the Fc or reducing the steric hindrance on antibodies constant region of other IgG molecules was interacting with antigens. developed. The binding of this anti-IgG antibody (also known as antihuman globulin or Coomb’s Examples are ficin, papain, bromelin, and trypsin reagent) results in the formation of a bridge between red cells that have IgG bound to their 💡 surface. This in turn creates agglutinates that are ADDITIONAL INFO visible to the human eye. AGITATION/CENTRIFUGATION NOTE: The IgG is a monomer in which it is smaller than the IgM ➔ The cells are usually repelling each other because which is pentameric. Therefore, the space that the IgM of zeta potential making them negatively charged covers is wider. Meaning if IgG antibody is used, the cells and repel each other. Through agitation and need to be closer to one another for them to attach and centrifugation, the cells are moved closer to one form a lattice formation. Meanwhile in IgM antibodies, it is another to promote agglutination. relatively easier for it to form a lattice formation since it has ➔ When using tubes, we use a centrifuge. But when a wider range of reach. Anti-antibody or the anti IgG using glass slides, we can either use our hands antibodies helps the IgG in the agglutination process. (by rotating the slide manually) or we can use a Anti-human globulin will be the one that will bridge the gap machine that vibrates to make the solution between the two antigen-antibody complexes to now form move. We are using the centrifugation process to the lattice formation. IgG can be used either with or without provide a force to the antigen and antibody to the AHG. We can still agitate or centrifuge in order for them bring them close to each other, And once they to agglutinate. But having AHG will help hasten the process. are near or close to each other it will be easier for the antibody to bind to the antigen. POLYMERS ➔ By decreasing the distance between two neighboring cells then we can form a cross link and observe visible agglutination. ANTIBODY ISOTYPE ➔ Zeta potential serves as a barrier between two red cells; keeps the two cells distant from one another. ➔ Reduced zeta potential decreases the gap between cells, making it easy for the binding of ➔ Given the pentad structure of IgM and its large 35 nm diameter, it can naturally cover the distance Immunology and Serology Laboratory | Trans by: (Bayotas, S., Javier, M.K., Letsoncito, K.J.)  MLS 17a IMMUNOLOGY AND SEROLOGY LABORATORY MODULE 3: Secondary Serologic Reactions: Unlabeled 2ND SEMESTER PRELIMS A.Y. 2023-2024 Immunoassay and Complement Fixation Test LECTURER: Matthew Tubola & Jeff Tolentino IgG antibodies. Therefore, lattice formation will SLIDE METHOD be faster. ➔ Albumin and polyethylene glycol (PEG) are both potentiators. ZETA POTENTIAL ➔ It is the natural repulsion of two RBCs because of its negative charge. ➔ Zeta potential is proportional to the ionic strength. The HIGHER the IONIC STRENGTH, the HIGHER the zeta potential. Considerations for the Slide method: Time ➔ Observe agglutination reaction while manually The WEAKER the IONIC STRENGTH, the WEAKER the zeta rotating the slides (rotation is done continuously potential. within the certain period of observation time). This makes the precipitate more visible, PROTEOLYTIC ENZYMES especially in 1+ grading, where there are less ➔ Examples are ficin, papain, bromelin, and trypsin ➔ visible or differentiable precipitates present and they are the most commonly used enzymes. (difficult to differentiate from Negative). ➔ These proteolytic enzymes are used to destroy the sialic acid present in the RBCs. Sialic acid in the RBCs provides the negative charge that GRADING OF AGGLUTINATION allows the zeta potential. If no negative charge is present, zeta potential decreases, and RBCs are TUBE METHOD SLIDE METHOD closer to each other and allow IgG to bind to the neighboring cell. 1 SOLID CLUMP VISIBLE 4+ 3+ AGGLUTINATION Table: Grading of Agglutination Reaction SEVERAL LARGE CLUMPS 3+ SLIGHT 2+ AGGLUTINATION TUBE METHOD NUMEROUS SMALL 2+ GRAY COLOR 1+ CLUMPS BARELY DISCERNIBLE 1+ NO AGGLUTINATION NEGATIVE CLUMPS SMOOTH NEGATIVE SOLUTION/SUSPENSION After centrifugation, you’ll see a red cell button at the bottom of the TESTS FOR AGGLUTINATION tube, that is agglutinated. ➔ Presence of button - there is agglutination; POSITIVE. 1. DIRECT (ACTIVE) AGGLUTINATION ➔ Absence of button (remains liquid) - there is no a. Widal Test agglutination; NEGATIVE. b. ABO Typing The tube is shaken gently, to move the button and will prevent it c. Viral Hemagglutination from adhering at the bottom of the tube. 2. INDIRECT (PASSIVE) AGGLUTINATION a. ASOT (antistreptolysin O Test) b. Latex Agglutination for RF (Rheumatoid Factor) 3. REVERSE PASSIVE AGGLUTINATION a. Latex Agglutination of CRP (C-Reactive Proteins) 4. COAGGLUTINATION 5. COOMB’S TEST (ANTIGLOBULIN-MEDIATED AGGLUTINATION) a. DAT (Direct Antiglobulin Test) b. IAT (Indirect Antiglobulin Test) 6. AGGLUTINATION INHIBITION Immunology and Serology Laboratory | Trans by: (Bayotas, S., Javier, M.K., Letsoncito, K.J.)  MLS 17a IMMUNOLOGY AND SEROLOGY LABORATORY MODULE 3: Secondary Serologic Reactions: Unlabeled 2ND SEMESTER PRELIMS A.Y. 2023-2024 Immunoassay and Complement Fixation Test LECTURER: Matthew Tubola & Jeff Tolentino a. Latex Particle Agglutination Inhibition (Classic text for hCG) b. Viral Hemagglutination Inhibition DIRECT AGGLUTINATION TEST ❖ Agglutination reactions where the antigens are found naturally on a particle. - RBC antigens - Bacterial antigens - Fungal antigens ❖ Direct bacterial agglutination uses whole pathogens as a source of antigen. ❖ The binding of antibodies to surface antigens on the PRINCIPLE: bacteria results in visible clumps ❖ Patients infected with Salmonella produce ❖ Widal test, ABO typing, and Viral hemagglutination antibodies against the antigens of the organism. are examples of Direct Agglutination Tests. Antibodies in serum, produced in response to exposure to Salmonella organisms will agglutinate bacterial suspension which carries homologous antigens. Conducted in 2 ways: Slide agglutination Widal Test ➔ Qualitative Slide Test ➔ Quantitative Slide Test Tube agglutination Widal Test 💡 ADDITIONAL INFO 💡 ADDITIONAL INFO DIRECT (ACTIVE) AGGLUTINATION Nowadays, it is not usually used because there are more ➔ Occurs when antigen (red blood cells, bacteria, specific tests for typhoid fever like the Typhidot test and fungi) is found naturally on a particle Typhi rapid. In the Widal Test, we are not only using ➔ Patient’s serum is diluted (in tubes or wells) and antigens from Salmonella typhi but also from Salmonella reacted with bacterial antigens specific for the paratyphi. suspected disease ➔ Tube agglutination has more accuracy than the ➔ Detection of antibodies is primarily used in the slide agglutination method. diagnosis of diseases for which the bacterial ➔ Slide agglutination is faster while tube agents are extremely difficult to cultivate. agglutination method takes time if performed NOTE: Widal Test - used for the detection of typhoid fever. The PREPARATION OF ANTIGENS: reagent used for this test is salmonella bacteria. Antigen’s suspensions may be prepared from suitable stock cultures in the laboratory, but generally commercially prepared ABO typing - red cell has a naturally occurring A and B suspensions are used. antigen 1. Salmonella typhi is used to prepare S. typhi O and S. typhi H antigens 2. Salmonella paratyphi A is used to prepare S. WIDAL TEST paratyphi A H antigens ❖ The Widal test is one method that may be used to 3. Salmonella paratyphi B is used to prepare S. help make a presumptive diagnosis of enteric fever, paratyphi B H antigens also known as typhoid fever. 4. O antigens for S. paratyphi A and B are not taken as ❖ The test was based on demonstrating the presence they cross-react with S. typhi O antigen of agglutinin (antibody) in the serum of an infected patient, against the H (flagellar) and O (somatic) antigens found in the cell wall of Salmonella typhi 💡 ADDITIONAL INFO and S. paratyphi. Four Reagents in Widal Test: Reagent O, H, AH, and BH ➔ Flagellar antigen is used to differentiate Salmonella typhi and Salmonella paratyphi Immunology and Serology Laboratory | Trans by: (Bayotas, S., Javier, M.K., Letsoncito, K.J.)  MLS 17a IMMUNOLOGY AND SEROLOGY LABORATORY MODULE 3: Secondary Serologic Reactions: Unlabeled 2ND SEMESTER PRELIMS A.Y. 2023-2024 Immunoassay and Complement Fixation Test LECTURER: Matthew Tubola & Jeff Tolentino ➔ O antigen is always positive if infected by Salmonella regardless of the type of Salmonella (causative agent) ➔ Agglutination with H antigen - Salmonella typhi ➔ Agglutination with AH antigen - Salmonella paratyphi A ➔ Agglutination with BH antigen - Salmonella paratyphi B Qualitative Widal Slide Test Procedure: 1. Label. Usually in Widal’s test there is a given test 💡 ADDITIONAL INFO card which already has circles like in the picture. Infecting pathogen: Salmonella typhi These circles are labeled with O, H, AH, BH, PC ➔ Positive result - presence of agglutination (positive control), and NC (negative control). ➔ O and H are positive and AH and BH are negative. The controls are important to ensure that our reagent is working. PC well should be positive for agglutination and NC well should be negative for agglutination. ➔ If there is something wrong with the controls, then we have to repeat the test or change the reagents. *** IN PERFORMING SERIAL DILUTIONS, a titer equal to or 2. 1 drop of Patient’s serum + 50 uL of corresponding greater than 1:80 is considered clinically significant (normal Salmonella spp antigen value is less than 1:80 antibody titer) Quantitative Widal Slide Test ➔ This is performed for the samples which showed positive agglutination during the qualitative test. ➔ For example, in the qualitative test, only the O and H are positive, that’s why we are only going to use the O and H in the quantitative test. ➔ Performed if the doctor wants to know the amount of antibody present. Procedure: 1. Prepare a slide labeled with O and H or separate slides for O and H. The slides will have 5 circles for each positive result. 3. Mix the serum and reagent with a separate applicator stick and spread well to fill the whole of the individual circle. 4. Rotate for 1 minute using a mechanical rotator or by 2. The 5 circles are labeled with 1:20, 1:40, 1:80, gently rocking each slide back and forth. 1:160, and 1:320, respectively. 3. Using a pipet, dispense 80uL, 40uL, 20uL, 10uL, and 5uL of undiluted serum to each well or circles of each O and H, respectively. This is because these are the antigens that showed positive agglutination from our previous samples. Immunology and Serology Laboratory | Trans by: (Bayotas, S., Javier, M.K., Letsoncito, K.J.)  MLS 17a IMMUNOLOGY AND SEROLOGY LABORATORY MODULE 3: Secondary Serologic Reactions: Unlabeled 2ND SEMESTER PRELIMS A.Y. 2023-2024 Immunoassay and Complement Fixation Test LECTURER: Matthew Tubola & Jeff Tolentino *** NOTE: Antibody titer greater than 1:80 is considered as significant infection and low titers indicate absence of infection. ABO TYPING Hemagglutination – an agglutination reaction that involves the red blood cells ➔ Best example is ABO blood group typing of human red blood cells 4. Add to each circle, a drop of antigen which showed an agglutination with the test sample on the qualitative method. O antigen should be added only to the O wells and the same goes with the H wells added with only H antigens. Reagents used: Antisera of IgM type can be used to detect the absence and presence of A and B antigens usually performed at 5. Using separate stirrers, uniformly mix the solution room temperature. over the circles. 6. Rotate for 1 minute using a mechanical rotator or VIRAL HEMAGGLUTINATION by gently rocking each slide back and forth. 7. Observe the presence of agglutination, interpret, Principle: Direct hemagglutination test uses the ability of some and report the titer. viruses to bind red blood cells, resulting in agglutination. 8. Titer of the patient serum using Widal test antigen suspension is the highest dilution of the serum sample that gives a visible agglutination. Tube agglutination Widal Test Procedure: 1. Bring all reagents at RT and mix well. 2. Prepare 4 sets of test tubes for individual antigens. Each set contains 1-8 test tubes. 3. To one set add 50 uL of corresponding antigens of Salmonella spp. 4. Mix well, cover and incubate these tubes overnight at 37 degrees Celsius (APPROX 18 HOURS) 5. After incubation, dislodge the sediments and observe for agglutination. Grading of hemagglutination: 6. Interpretation of results: the antibody titer of the Tube method test sample is its highest dilution that gives a visible Negative – smooth suspension reaction. 1+ - Barely discernable clumps 2+ - Numerous smaller clumps 3+ - Several large clumps 4+ - One solid clump Slide method Strong agglutination – large clumps and clear background Weak agglutination – small clumps and cloudy background Negative – even suspension and cloudy background Immunology and Serology Laboratory | Trans by: (Bayotas, S., Javier, M.K., Letsoncito, K.J.)  MLS 17a IMMUNOLOGY AND SEROLOGY LABORATORY MODULE 3: Secondary Serologic Reactions: Unlabeled 2ND SEMESTER PRELIMS A.Y. 2023-2024 Immunoassay and Complement Fixation Test LECTURER: Matthew Tubola & Jeff Tolentino ❖ Rheumatoid factor is an IgM autoantibodies reacting INDIRECT AGGLUTINATION TEST with the Fc portion of IgG molecules ❖ Passive Agglutination - Otherwise known as Indirect Agglutination Principle: Latex particles are coated with human gamma ❖ Test to detect the presence of specific antibody in globulins. When the reagent is mixed with serum containing which inert particles or cells with no foreign Rheumatoid factor (RF), the particles will agglutinate, which is antigenic markers are coated with known soluble interpreted as a POSITIVE reaction antigen and mixed with serum. ❖ It employs particles that are coated with antigens not normally found in the surface including red blood cells, latex, gelatin and silicates. ❖ Soluble antigens passively adsorbed or chemically coupled to red blood cells or inert particles (latex, gelatin, silicates, charcoal, bentonite) ❖ This is typically precipitation converted to agglutination by increasing size of “antigen” particles. ❖ The use of beads or particles provides the advantage POSITIVE: Presence of agglutination (Reported as of CONSISTENCY, UNIFORMITY AND STABILITY. GREATER THAN OR EQUAL TO 8 IU/mL) ❖ Reactions is easy to red visually and give quick NEGATIVE: Absence of agglutination (Reported as results LESS THAN 8 IU/mL) REVERSE PASSIVE AGGLUTINATION ❖ The antibody must be oriented in a way that the active site is facing outward ❖ This is used for the detection of microbial antigens such as Group A and B Streptococci, Staphylococcus aureus, Neisseria meningitidis etc. ❖ This is also used to measure levels of certain Examples: Antistreptolysin O Titer (ASOT), Latex Agglutination therapeutic drugs, hormones and plasma proteins. test for Rheumatoid Factor, Monospot Latex Agglutination for Example: C-Reactive Protein (CRP) Infectious Mononucleosis. LATEX AGGLUTINATION FOR C-REACTIVE PROTEIN ANTISTREPTOLYSIN O TEST Principle: The latex particles used in the CRP latex agglutination Principle: Latex particles are coated with streptolysin O antigen. test are coated with anti-human CRP that agglutinate upon When the reagent is mixed with serum containing mixing with patient serum containing CRP. anti-streptolysin O (ASO), the particles will agglutinate, which is interpreted as a POSITIVE reaction POSITIVE: Presence of agglutination (Reported as GREATER THAN OR EQUAL TO 0.6 mg/dL) POSITIVE: Presence of agglutination (Reported as NEGATIVE: Absence of agglutination (Reported as GREATER THAN OR EQUAL TO 200 uL) LESS THAN 0.6 mg/dL) NEGATIVE: Absence of agglutination (Reported as LESS THAN 200 uL) COAGGLUTINATION Procedure: Add latex reagent to patient’s serum and rotate the ❖ Protein ‘A’, produced by Staphylococcus aureus slide for 3 minutes. Observe for presence of agglutination Cowan-I strain has high affinity for IgG antibodies of (Procedure varies according to reagent insert) certain species. Such bacterial cells can be coated with specific antiviral antibodies, which bind via LATEX AGGLUTINATION FOR RF their Fc portion to the protein A molecules, leaving the Fab region free. Immunology and Serology Laboratory | Trans by: (Bayotas, S., Javier, M.K., Letsoncito, K.J.)  MLS 17a IMMUNOLOGY AND SEROLOGY LABORATORY MODULE 3: Secondary Serologic Reactions: Unlabeled 2ND SEMESTER PRELIMS A.Y. 2023-2024 Immunoassay and Complement Fixation Test LECTURER: Matthew Tubola & Jeff Tolentino ❖ Staphylococcus aureus Cowan-I strains are more preventing their binding to and agglutination of the stable, economical and resistant to ionic changes particles. than latex beads. ❖ Absence of agglutination is a positive reaction ❖ The co-agglutination reaction is highly specific but ❖ Common tests following this principle are may not be as sensitive for detecting small Latex Particle Agglutination Inhibition quantities of antigen as latex agglutination. (Classic test for hCG) ❖ In a positive test, protein A bearing S. aureus coated Viral Hemagglutination Inhibition with antibodies will be co-agglutinated if mixed with ❖ Red blood cells have naturally occurring viral specific antigen. receptors ❖ When virus is present, spontaneous agglutination COOMB’S TEST occurs, because the virus particles link the RBCs ❖ Otherwise known as Antiglobulin-Mediated together Agglutination ❖ Presence of patients antibody inhibits the ❖ Anti-human globulin (AHG) contains an IgG with agglutination reaction specificity for the constant region of other IgG LATEX PARTICLE AGGLUTINATION INHIBITION (CLASSIC antibodies. HCG TEST) ❖ When the binding occurs, a bridge is created within the IgG-bound red cells, causing them to be visibly ❖ Not commonly used nowadays because of the more agglutinated. rapid test for hCG which uses the lateral flow ❖ Direct and Indirect Coombs test – both tests involve immunoassay IgG and anti human immunoglobulin-mediated hemagglutination ❖ Anti-human globulin (AHG reagent) Color of reagent is GREEN ❖ Used to detect human antibodies in a number of reactions in the serology lab. ❖ Anti-human immunoglobulin is an antibody that is made in other species and binds to the Fc region of human immunoglobulin ❖ Pregnant: DIRECT COOMBS TEST The anti-hCG added will neutralize the ❖ Measures when an antibody is present on the hCG in the urine and they will form an individual’s red blood cells antigen antibody complex. ❖ Detects IN VIVO sensitization of antibodies to red There is no more room for the hCG cells antiserum to bind to the second reagent ❖ Patient RBCs are washed with saline and mixed with (hCG-coated latex). AHG reagent at room temperature, then No visible agglutination because all of centrifuged, resuspended and examined for the hCG antisera has reacted with the agglutination. hCG that is present in the urine of the ❖ Used to detect autoimmune hemolytic anemia, pregnant woman. No visible hemolytic transfusion reaction and hemolytic agglutination will be considered as a disease of the newborn. positive result. INDIRECT COOMBS TEST ❖ Measures when an antibody is present on the patient’s red blood cells ❖ To demonstrate presence of antibody or complement in patient serum, which results in the IN VITRO coating of RBCs by the antibody or complement component ❖ Used for weak D testing ❖ Not pregnant: Same principle is used but the urine does AGGLUTINATION INHIBITION not contain hCG which means there will ❖ Based on the competition between particulate and be no binding that will occur. soluble antigens for limited antibody-combining Upon addition of the hCG-coated latex sites particles, they will bind to the antibodies ❖ Agglutination inhibition or hemagglutination and will result in a visible agglutination inhibition is the inhibition of these reactions, Immunology and Serology Laboratory | Trans by: (Bayotas, S., Javier, M.K., Letsoncito, K.J.)  MLS 17a IMMUNOLOGY AND SEROLOGY LABORATORY MODULE 3: Secondary Serologic Reactions: Unlabeled 2ND SEMESTER PRELIMS A.Y. 2023-2024 Immunoassay and Complement Fixation Test LECTURER: Matthew Tubola & Jeff Tolentino since the binding sites are open because of the absence of hCG in the urine. No hCG in the urine that the hCG antiserum will bind to, hence there is no antigen-antibody binding that will happen, so no neutralization. When hCG-coated latex particles are added, this will react with the hCG antiserum and therefore produce a visible agglutination. ❖ Negative Reaction (With agglutination): Visible agglutination will be considered If there is no viral antibody, when the as a negative result serum is added to the virus reagent there will be no binding that will occur which allows the binding between the red blood cell and the virus forming the visible hemagglutination. Presence of agglutination is considered as a negative result ❖ PROCEDURE: No agglutination = POSITIVE Presence of agglutination = NEGATIVE Done on microtiter wells VIRAL HEMAGGLUTINATION INHIBITION Performed through serial dilution ❖ This hemagglutination inhibition (HI) assay is used to titrate the antibody response to a viral infection. PART 2: ❖ This HI assay takes advantage of some viruses' ability PRECIPITATION to hemagglutinate (bind) red blood cells, therefore ❖ Requires formation of a visible lattice that results forming a “lattice” and preventing the red blood from a combination of soluble antigen to its soluble cells from clumping since nucleic acids of viruses antibody. encode proteins, such as hemagglutinin, that are ❖ Usually works best when Ag and Ab are at optimal expressed on the surface of the virus. proportions (zone of equivalence). ❖ Red blood cells have naturally occurring viral ❖ Precipitation occurs in 2 steps: receptors Rapid invisible formation of Ag/ Ab ❖ When a virus is present, spontaneous agglutination aggregates occurs because the virus particles link the RBCs Slow-forming visible complex lattice of together. interlocking aggregates ❖ Presence of patients’ antibody inhibits the agglutination reaction. PRECIPITATION CURVE ❖ PROZONE - Antibodies are in excess, so there are insufficient reactive sites on antigen for the lattice formation. ❖ ZONE OF EQUIVALENCE - The concentration and ratio of both antigen and antibody are at equilibrium. The ideal zone to have visible precipitation. Ag and Ab concentrations at equilibrium. ❖ POST-ZONE - Antigens are in excess, so there are ❖ Positive Reaction (No agglutination): insufficient reactive sites on antibodies for lattice If the antibody binds to the virus and formation. then we add the red cells, there will be no agglutination. The absence of agglutination is considered a positive result since we are looking for the viral antibody in the serum of the patient. The virus and red blood cells are considered as reagents Immunology and Serology Laboratory | Trans by: (Bayotas, S., Javier, M.K., Letsoncito, K.J.)  MLS 17a IMMUNOLOGY AND SEROLOGY LABORATORY MODULE 3: Secondary Serologic Reactions: Unlabeled 2ND SEMESTER PRELIMS A.Y. 2023-2024 Immunoassay and Complement Fixation Test LECTURER: Matthew Tubola & Jeff Tolentino TESTS FOR PRECIPITATION Ag or Ab diffuses (1 REACTANT = SINGLE) 1. Measure of Precipitation in SOLUTION Single means ikaw, ay, single a. Ring Precipitation Test means the reactant that b. Flocculation Test diffuses is only one. i. Rapid Plasma Reagin (RPR) We have wells within the gel. 2. Instrumental Methods to Enhance Sensitivity in The gel is incorporated with Measuring Precipitation in Solution antibodies, then we add a. Turbidimetry antigen on the well, the b. Nephelometry antigen will then diffuse. 3. Precipitation by PASSIVE IMMUNODIFFUSION Double Linear Immunodiffusion a. Linear Immunodiffusion (One Both Ab and Ag are moving (2 Dimensional) REACTANTS = DOUBLE) i. Single Linear Immunodiffusion Within the gel we have two (Oudin technique) (2) wells, one for each ii. Double Linear reactant. No reactant is Immunodiffusion (Oakley incorporated in the gel. Fulthorpe immunodiffusion) Then we add antigen and b. Radial Immunodiffusion (Two antibody to their respective Dimensional) wells, and both of them i. Single Radial Immunodiffusion diffuse in the gel, that is why (Mancini Technique and Fahey double immunodiffusion. Technique) The result here is linear. ii. Double Radial Immunodiffusion RADIALIMMUNODIFFUSION (Ouchterlony Technique) 4. Precipitation by ELECTROPHORETIC TECHNIQUES ❖ Same principle with linear, however, the reaction a. Electroimmunodiffusion (precipitation) is in CIRCULAR OR RADIAL form i. One Dimension Electroimmunodiffusion PRECIPITATION BY ELECTROPHORETIC TECHNIQUES 1. Single Electroimmunodiff ❖ Passive immunodiffusion + Electric current usion (Rocket ❖ Faster than passive since we incorporate electric Immunoelectropho current, which enhances diffusion of reactants. resis) ❖ One-dimension electroimmunodiffusion 2. Double One (1) gel Electroimmunodiff Single = one (1) reactant diffuses usion (Counter Double = two (2) reactants diffuse Immunoelectropho ❖ Two-dimension electroimmunodiffusion resis) Two (2) separate gels ii. Two Dimension ❖ Immunoelectrophoresis Electroimmunodiffusion Replaced by immunofixation 1. Cross electrophoresis due to difficulty in Immunoelectropho interpretation. resis ❖ Immunofixation electrophoresis b. Immunoelectrophoresis Easier to interpret compared to immunoelectrophoresis TESTS FOR PRECIPITATION MEASUREMENT OF PRECIPITATION IN SOLUTION PRECIPITATION BY PASSIVE IMMUNODIFFUSION RING PRECIPITATION TEST LINEAR IMMUNODIFFUSION ❖ The ring precipitation test is usually performed in a ❖ We have Ag and Ab wells, we add Ag and Ab in their tube and requires the formation of antigen-antibody respective wells, and the antigens will diffuse in the precipitate between two fluids as a ring of gel and the antibody also diffuses in the well. precipitate that is layered on the surface of the ❖ If their concentrations are at the zone of antibody. equivalence, we see a LINEAR formation of FLOCCULATION TEST precipitation (linear immunodiffusion). ❖ It can be single or double linear immunodiffusion. ❖ Flocculation tests may be performed in a slide or 1. Single Linear Immunodiffusion tube. In this test, a drop of antigen solution is added Immunology and Serology Laboratory | Trans by: (Bayotas, S., Javier, M.K., Letsoncito, K.J.)  MLS 17a IMMUNOLOGY AND SEROLOGY LABORATORY MODULE 3: Secondary Serologic Reactions: Unlabeled 2ND SEMESTER PRELIMS A.Y. 2023-2024 Immunoassay and Complement Fixation Test LECTURER: Matthew Tubola & Jeff Tolentino to a drop of the patient’s serum on a slide and the result is recorded after shaking the slide. ❖ In a positive test, the floccules appear which can be seen by the naked eye or demonstrated well under the microscope. ❖ E.g., RPR test for syphilis (RPR Card) and VDRL test for syphilis (slide) PRINCIPLE: The test card used is white in color because charcoal (carrier) is black. Add 1 drop of undiluted serum, spread, and then add one drop of reagent antigen. Rotate for 8 MINUTES on the mechanical rotor at 100 RPM. Read results while in a wet state under a high-intensity incandescent lamp or strong daylight. ○ When dry, it shows a grainy appearance and may be interpreted as false-positive. FLOCCULATION TEST a. Rapid plasma reagin (RPR) ❖ The RPR is a nontreponemal serological test for syphilis that detects reagin, an antibody formed against cardiolipin during the progress of the Positive = Presence of floccules disease. Negative = Absence of floccules ❖ The reagent antigen consists of: In RPR, report as REACTIVE or NON-REACTIVE Cardiolipin - the antigen Lecithin INSTRUMENTAL METHODS TO ENHANCE SENSITIVITY IN Cholesterol MEASURING PRECIPITATION IN SOLUTION 10% Choline chloride (inactive complement)- provides stability to the ❖ When antibodies and antigens are mixed in reagent antigen. solution, the lattice structures begin to form, and an EDTA (stabilizer) - anticoagulant initial cloudiness of the solution is followed by the Charcoal (visualizing reagent)l - inert lattice structures precipitating out of solution. particle where cardiolipin binds ❖ Two types of instrumentation: turbidimetry and nephelometry TURBIDIMETRY ❖ The initial cloudiness is measured by passing a light through the solution (in the cuvette) and determining the amount of light that comes directly across the solution into the detector. ❖ This is a measure of the amount of light that is lost due to scatter by the lattice structures. ❖ The amount of scatter is proportional to the concentration of the molecules in the lattice structures. Immunology and Serology Laboratory | Trans by: (Bayotas, S., Javier, M.K., Letsoncito, K.J.)  MLS 17a IMMUNOLOGY AND SEROLOGY LABORATORY MODULE 3: Secondary Serologic Reactions: Unlabeled 2ND SEMESTER PRELIMS A.Y. 2023-2024 Immunoassay and Complement Fixation Test LECTURER: Matthew Tubola & Jeff Tolentino NEPHELOMETRY ❖ Nephelometry is similar to turbidimetry but does not measure the light directly across from the light source but at a 10° to 90° (70° typically used) angle from the light source. DOUBLE LINEAR IMMUNODIFFUSION AKA OAKLEYFULTHORPE DIFFUSION TECHNIQUE ❖ Antibody is incorporated in a gel, above which a column of plain agar. ❖ The antigen is layered on top of the agar medium. ❖ The antigens and antibodies move towards each other through the intervening column of plain agar. This will result in the formation of a precipitate when it meets at optimum PRECIPITATION BY PASSIVE IMMUNODIFFUSION proportion. ❖ This method allows the combination of specific antigens and antibodies in a semi-solid gel or media. ❖ This is performed by using agar or agarose gel, which stabilizes the diffusion process and allows visualization of precipitin bands. ❖ This type of immunodiffusion does not use an electric current to diffuse. ❖ Immunodiffusion reactions are classified according to: Number of reactants diffusing (Single or Double) SINGLE RADIAL IMMUNODIFFUSION Direction of Diffusion (One Dimensional or Two Dimensional) ❖ In this type of immunodiffusion, the antibodies are spread homogeneously in an agar medium, and the antigens diffuse within the agar. ❖ Here, the gel agar contains diluted antibodies specific to certain antigens. ❖ A ring of precipitate forms upon the addition of the antigens into the well, which is a cut in the agar. ❖ The ring forms at the equivalent point between the antibodies and the antigens. ❖ The diameter of the ring depends on the antigen concentration; thus, the higher the antigen concentration, the greater the diameter of the ring. SINGLE LINEAR IMMUNODIFFUSION AKA Oudin Diffusion Technique ❖ The antibody is incorporated in an agar gel in a test tube and then the antigen is layered over it. The antigen diffuses downwards through the gel, forming a line of precipitation. ❖ Precipitate dissolves as the concentration of the antigen increases. ❖ The number of bands indicates the number of different antigens. Immunology and Serology Laboratory | Trans by: (Bayotas, S., Javier, M.K., Letsoncito, K.J.)  MLS 17a IMMUNOLOGY AND SEROLOGY LABORATORY MODULE 3: Secondary Serologic Reactions: Unlabeled 2ND SEMESTER PRELIMS A.Y. 2023-2024 Immunoassay and Complement Fixation Test LECTURER: Matthew Tubola & Jeff Tolentino ❖ In this method, both antigens and antibodies diffuse independently through agar gel in 2 dimensions, horizontally and vertically. ❖ As they diffuse radially, their concentration diminishes. At the point in the diffusion where the relative concentrations of antibody and antigen reach equivalence, a precipitin line appears. ❖ The technique is named for Örjan Ouchterlony, the Swedish physician who invented the test in 1948. PRINCIPLE: The test is performed by cutting wells in the agar gel ❖ Actual appearance of our single radial poured on a glass slide or in a petri dish. The antibodies are immunodiffusion. placed in a center well and different antigens are added to the ❖ Antibody is spread over the agar. well surrounding the center well. After an incubation period of ❖ Well is made at the center and antigen is placed, 12-48 hours in a moisture chamber, the precipitin lines are which will move and diffuse horizontally and formed at the site of the combination of antigen and antibody. vertically. ❖ At the time where the maximum amount of antigen-antibody complex is reached, it will form a lattice, producing a precipitin ring (they have met the zone of equivalence). ❖ The diameter of the ring that will be formed is directly proportional to the amount of antigen present in the sample. PATTERNS OF RESULTS ❖ Absence of precipitin ring is a NEGATIVE result. 1. PARTIAL IDENTITY ❖ There are 2 methods: ❖ Fusion of 2 lines with spur. Endpoint Method – Mancini Technique ❖ Interpretation: Partial Identity or there is Kinetic Method – Fahey-McKelvey cross-reaction. Technique ❖ The spur formed always points to the simpler MANCINI TECHNIQUE Uses measurements taken antigen. after the endpoint of ❖ Two antigen wells share a common epitope, which is equivalence is reached. why antibodies are bound to form a precipitate, although part of that has also other different Occurs between ○ 24-48 epitopes. Therefore, as you can see, there is a line hours (IgG) ○ 72 hours (IgM) that is formed for that other epitope. The square of the diameter is directly proportional to the concentration of the antigen. FAHEY-MCKELVEY Uses measurements taken TECHNIQUE before the endpoint of equivalence is reached. B. PARTIAL NON-IDENTITY Readings are taken at ❖ Presence of crossed lines. about 18 hours. ❖ Interpretation: Demonstrates 2 separate reactions or the compared antigens shared no common The diameter of the ring epitopes. is proportional to the log of ❖ There are 2 separate epitopes to separate antigens. the antigen concentration So, different antibodies will bind to each of them, forming their own lines. As observed, the lines DOUBLE RADIAL IMMUNODIFFUSION formed to cross each other, indicating Non-identity. ❖ Also known as OUCHTERLONY (Double Angular Immunodiffusion). Immunology and Serology Laboratory | Trans by: (Bayotas, S., Javier, M.K., Letsoncito, K.J.)  MLS 17a IMMUNOLOGY AND SEROLOGY LABORATORY MODULE 3: Secondary Serologic Reactions: Unlabeled 2ND SEMESTER PRELIMS A.Y. 2023-2024 Immunoassay and Complement Fixation Test LECTURER: Matthew Tubola & Jeff Tolentino Immunofixation Electrophoresis PROCEDURE: 1. The source of antigen is serum, which is electrophoresed to separate out the main proteins. 2. A trough is then cut in the gel parallel to the line of separation. 3. Antiserum is placed in the trough and the gel is incubated for 18 to 24 hours. 4. Double diffusion occurs at right angles to the electrophoretic separation and precipitin lines develop where a specific antigen-antibody C. IDENTITY combination takes place. ❖ Fusion of lines at their junction to form an arc. Interpretation is difficult; therefore, it has largely been replaced ❖ Interpretation: Serologic identity or presence of by immunofixation electrophoresis, which gives faster results common epitope (two identical antigens). and is easier to interpret. PRECIPITATION BY ELECTROPHORETIC TECHNIQUES ELECTROPHORESIS ❖ A method that separates molecules according to differences in their electric charge when they are placed in an electric field. A direct current is forced through the gel, causing antigen, antibody, or both to migrate. IMMUNOELECTROPHORESIS: ❖ Process that combines immunodiffusion and electrophoresis. ❖ A double-diffusion technique that incorporates electrophoresis to enhance results. ❖ E.g., One Dimensional Immunoelectrophoresis Rocket Immunoelectrophoresis Countercurrent Immunoelectrophoresis Two Dimensional Immunoelectrophoresis Crossed Immunoelectrophoresis Immunoelectrophoresis Immunology and Serology Laboratory | Trans by: (Bayotas, S., Javier, M.K., Letsoncito, K.J.)  MLS 17a IMMUNOLOGY AND SEROLOGY LABORATORY MODULE 3: Secondary Serologic Reactions: Unlabeled 2ND SEMESTER PRELIMS A.Y. 2023-2024 Immunoassay and Complement Fixation Test LECTURER: Matthew Tubola & Jeff Tolentino ❖ The antibodies are in the gel. ❖ The sample which contains the antigen is added to IMMUNOELECTROPHORESIS the wells. ❖ The machine is attached to the power which ONE-DIMENSIONAL IMMUNOELECTROPHORESIS: provides the electric current to the antigen. ROCKET IMMUNOELECTROPHORESIS ❖ The antigen will move from the well to the gel and it ❖ Also known as Laurell Technique will form a precipitin. Immunoelectrophoresis. ❖ The shape of the precipitin is either a rocket or ❖ A modification of single radial immunodiffusion bullet shape. using electrophoresis, developed by Laurell. ONE-DIMENTIONAL IMMUNOELECTROPHORESIS: ❖ It is called so due to the appearance of the precipitin COUNTERCURRENT IMMUNOELECTROPHORESIS bands in the shape of a cone-like structure (rocket ❖ This method is the Ouchterlony technique enhanced appearance) at the end of the reaction. and made quicker by using an electric current to ❖ The height of the rocket precipitin is directly bring the antigen and antibody together. proportional to the amount of antigen in the ❖ Both Ag and Ab are placed in wells in an agar gel and sample. made to migrate toward each other by ❖ Mainly used for the quantitative estimation of the electrophoresis. antigen in the serum. ❖ The pH of the buffer is so chosen (8.6- Barbital PRINCIPLE: Buffer) that the Ab is positively charged, and the Ag Antibody is incorporated in the gel and antigen is is negatively charged. placed in wells cut in the gel. Electric current is then passed PRINCIPLE: through the gel, (a negatively charged antigen is Ag is placed in the cathodic well (negative end) and electrophoresed in a gel-containing antibody which facilitates antiserum is placed in the anodic well (positive end). Ag will the) which facilitates the migration of the antigen into agar. This migrate towards the anode while Ab migrates towards the results in the formation of the precipitin line which has the cathode and precipitation occurs at equivalence within 30 to 60 shape of a rocket. The height of the rocket, measured from the minutes. If there is no precipitin line, the result is negative. well to the apex, is directly proportional to the amount of Otherwise, the result is positive. The antigen is placed on the antigen in the sample. cathode side while the antibody is placed on the anode side. They will both migrate towards the middle where the precipitin line will form which can be considered as the zone of equivalence. The advantage is that in a couple of minutes or in an hour, we can already obtain results. IMMUNOELECTROPHORESIS TWO-DIMENSIONAL IMMUNOELECTROPHORESIS: Crossed Immunoelectrophoresis Immunology and Serology Laboratory | Trans by: (Bayotas, S., Javier, M.K., Letsoncito, K.J.)  MLS 17a IMMUNOLOGY AND SEROLOGY LABORATORY MODULE 3: Secondary Serologic Reactions: Unlabeled 2ND SEMESTER PRELIMS A.Y. 2023-2024 Immunoassay and Complement Fixation Test LECTURER: Matthew Tubola & Jeff Tolentino ❖ Two-dimensional immunoelectrophoresis, also ❖ After incubation, the gel is now washed, pressed, known as Cross Immunoelectrophoresis, is a variant dried, and stained. of rocket electrophoresis. ❖ Patterns are then examined. ❖ Has 2 stages: First dimension and Second dimension. FIRST DIMENSION COMPLEMENT FIXATION ❖ Antigens in solution are separated by ❖ Based on the fact that when antigen combines with electrophoresis. an antibody in the presence of complement, the ❖ Without antibodies. complement is fixed by the antigen-antibody SECOND DIMENSION complex and is unable to react with cells sensitized with other antigen-antibody complexes. ❖ Electrophoresis is carried out again, but ❖ RBCs sensitized with specific antibodies are used as perpendicular to that of the first stage to obtain an indicator of the presence of an “unfixed” rocket-like precipitation. complement. ❖ Incorporated with antibodies. ❖ Lysis of these erythrocytes indicates the presence of unfixed complement ❖ Lack of hemolysis indicates that the antigen-antibody reaction fixes the complement. IMMUNOFIXATION ELECTROPHORESIS ❖ Similar to immunoelectrophoresis except that after electrophoresis takes place, antiserum is applied directly to the gel’s surface rather than placed in a trough. ❖ Agarose or cellulose acetate can be used for this purpose. ❖ Immunodiffusion takes place in a shorter time and results in a higher resolution than when an antibody diffuses from a trough. ❖ Because diffusion is only across the thickness of the gel, approximately 1mm, the reaction usually takes place in less than 1 hour. ❖ The pattern indicates an abnormality in the gamma region of the sample. ❖ This is monoclonal, but to know the specific answer, COMPLEMENT FIXATION STAGE you need to perform IMMUNOFIXATION. ❖ If there is a presence of antibodies i